| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Pediatrics, Division of Hematology/Oncology and
Department of Internal Medicine, Division of Nephrology, University of Michigan, Ann Arbor, MI 48109
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(PKC) and Raf-1 translocation from
cytosol to plasma membrane by >90%. Extracellular signal-regulated
protein kinase-1 and -2 (ERK1 and ERK2) phosphorylation was similarly
blocked. We also investigated phosphatidylinositide 3-kinase (PI
3-kinase) activity and Syk phosphorylation using piceatannol,
wortmannin, and LY294002, inhibitors of PI 3-kinase. The
phosphorylation of Syk preceded the activation of PI 3-kinase. Both
wortmannin and piceatannol inhibited PI 3-kinase, but only piceatannol
inhibited Syk. In contrast to piceatannol, wortmannin did not inhibit
PKC and Raf-1 translocation. To elucidate signaling downstream of
Syk activation, we assessed whether the cell-permeable diacylglycerol
analogue didecanoylglycerol could normalize PMN phagocytosis, PKC
and Raf-1 translocation, and ERK1 and ERK2 phosphorylation inhibited by
piceatannol. The addition of didecanoylglycerol to the Syk-inhibited
phagocytosing PMN normalized all three without a concomitant effect on
PI 3-kinase activity and Syk phosphorylation. We conclude that Syk
activation following Fc
receptor engagement initiates downstream
signaling events leading to mitogen-activated protein kinase activation
independent of PI 3-kinase activation. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
receptor on the cell
(1, 2, 3). PMNs possess three classes of Fc receptors,
inducible and constitutive, FcRI, FcRIIA, and FcRIIIB, all of
which are involved in phagocytosis (1, 4). Protein
tyrosine kinases have been implicated in signaling events initiated by
Fc receptors (5, 6). FcIIA receptors do not possess
intrinsic tyrosine kinase activity, but their cytoplasmic domains
possess sequences that facilitate interactions with protein tyrosine
kinases. Recently, the subunits of the cytoplasmic domain of the
FcIIA receptor have been implicated in signal transduction because
they include conserved tyrosine-containing sequences, which are thought
to bind to the SH2 (SRC homology 2) domain of protein tyrosine
kinases (7, 8).
Syk possesses two N-terminal SH2 domains that bind in tandem to sites
located within the immunoreceptor tyrosine-based activation motifs
(ITAMs) of Ag receptor subunit (9). Following Fc
receptor engagement in monocytes and macrophages, Syk is associated
with the -chain of FcRI and FcRIIIA and with the cytoplasmic
domain of FcRIIA, whereupon it becomes phosphorylated on tyrosine,
and is activated. Syk is recruited through its SH2 domains to the Fc
receptors, subsequently undergoes autophosphorylation, and induces the
phosphorylation of multiple substrates, including other Fc receptor
ITAMs and downstream effectors (10, 11). Upon transfection
with human Fc receptors, COS-1 cells acquire phagocytotic
properties, which in the case of FcRI and Fc RIIIA isoforms are
dependent on an ITAM within the -chain of the receptor
(12, 13, 14). However, reconstitution of the receptor complex
results in only marginal phagocytic activity, which can be
significantly increased by cotransfection with Syk (1).
Syk plays a major role in phagocytosis, because a chimeric
transmembrane protein containing only the Syk catalytic domain is
capable of triggering phagocytosis in COS cells (15).
Clustering of the FcRIII-Syk fusion in COS-1 cells results in a
phagocytic signal that is dependent on an intact Syk kinase domain
(15). Treatment of monocytes with Syk antisense
oligodeoxynucleotides has been reported to abrogate phagocytosis
(16). Although it is known that Syk phosphorylation is
involved in Fc receptor engagement, little is known concerning
either the mechanism or the identity of many of the downstream
tyrosine-phosphorylated substrates in EIgG-stimulated PMN undergoing
phagocytosis.
We have shown that the MAP kinase pathway is another key component in
the transduction of signals leading to Fc receptor-mediated
phagocytosis in PMN (17). This pathway consists of a
linear cascade of the protein kinases Raf-1, MEK, ERK1, and ERK2
(18, 19, 20). The ERK proteins are phosphorylated and
activated by the dual specificity kinase MEK, which is phosphorylated
and activated by the serine/threonine kinase Raf. We previously
reported that PKC is a key component of the phagocytic pathway and
is translocated from cytosol to plasma membrane in phagocytosing PMN.
This, in turn, leads to translocation of Raf-1 to the plasma membrane
followed by phosphorylation of ERK1 and ERK2 (21).
Phosphatidylinositide 3-kinase (PI 3-kinase) has also been implicated
previously in signaling by all three classes of Fc receptor
(1, 22). Activation of PI 3-kinase results in the
appearance of the lipid products of this enzyme, phosphatidylinositol
3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate
(23, 24). The PI 3-kinase is composed of a catalytic
subunit p110 and an associated regulatory subunit, p85 (25, 26). Wortmannin, an inhibitor of PI 3-kinase, binds to the
catalytic subunit of PI 3-kinase in PMN (27).
The purpose of the present study was to examine the requirement of PMN
Syk activation to initiate downstream signaling events during Fc
receptor-mediated phagocytosis. We evaluated whether Syk activation
would lead to PI 3-kinase activation and PKC and Raf-1
translocation, the latter translocations being associated with ERK
activation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Wortmannin and LY294002 were purchased from Biomol (Plymouth
Meeting, PA), and piceatannol
(3,4,3',5'-tetrahydroxy-trans-stilbene) was obtained from
Alexis (San Diego, CA); FMLP, cytochalasin B, and di-isopropyl
fluorophosphate were purchased from Sigma (St. Louis, MO). Polyclonal
Abs against ERK1 and ERK2 (p44/42) recognizing the phosphorylated form
of both p42 and p44 were obtained from New England BioLabs (Beverly,
MA). Polyclonal and monoclonal Ab against Syk and polyclonal Ab against
Hck and Fgr were obtained from Santa Cruz Biotechnology (Santa Cruz,
CA). Monoclonal anti-phosphotyrosine Ab 4G10 and polyclonal Ab
against PI 3-kinase p85 were purchased from Upstate Biotechnology (Lake
Placid, NY), and monoclonal Abs against PKC and Raf-1 were obtained
from Transduction Laboratories (Lexington, KY). The HRP-conjugated
sheep anti-mouse Abs were obtained from Amersham (Arlington
Heights, IL), and HRP-conjugated anti-rabbit Ab was obtained from
Santa Cruz Biotechnology. sn-1,2-dioctanoylglycerol (DiC8),
sn-1,2-didecanoylglycerol (DiC10), and
L-
-phosphatidylinositol (liver) were purchased
from Avanti Polar Lipids (Alabaster, AL). The
[-32P]ATP was obtained from ICN
Pharmaceuticals (Irvine, CA). The G-CSF was a gift from Amgen (Thousand
Oaks, CA).
Cells
Human PMN were isolated from human peripheral blood as described previously (28). Briefly, fresh whole blood was obtained by venipuncture from healthy volunteers and immediately added to acid citrate dextrose. The PMN were purified by dextran sedimentation followed by hypotonic lysis to remove the majority of erythrocytes and then centrifuged through Ficoll-Paque (Pharmacia LKB Biotechnology, Piscataway, NJ) to remove contaminating mononuclear cells. Before activation of cells and subcellular fractionation, the cells were incubated for 5 min on ice with 5 mM di-isopropyl fluorophosphate, washed, and resuspended in the desired buffer.
Phagocytic targets
Sheep erythrocytes were purchased from BioWhittaker (Walkersville, MD) and were opsonized with anti-sheep erythrocyte IgG (ICN Pharmaceuticals) as previously described (29).
Phagocytosis assay
The phagocytosis assay was conducted essentially as outlined by Pommier et al. (30). For studies with inhibitors, PMN were suspended at 2 x 106/ml in PBS containing 1 mM Ca2+ and 1 mM Mg2+ and were incubated with different concentrations of inhibitors for 30 min at 22°C. In other experiments PMN were preincubated with inhibitors and then treated with DiC10. Following the incubation, PMN underwent phagocytosis with EIgG (1 x 108/ml), and the incubation was continued for an additional 30 min at 37°C. EIgG that were not ingested were lysed with distilled water, and isotonicity was restored using KCl. Samples were fixed with glutaraldehyde and evaluated microscopically. The number of EIgG ingested per 100 PMN was determined. Inhibition of phagocytosis in the presence of inhibitors was expressed as a percentage of the control value, with control being phagocytosis by PMN in the absence of inhibitor treatment.
Immunoblotting
PMN lysates (1–2 x 106 PMN/sample
in 30–40 µl of buffer) were combined with sample buffer, boiled for
5 min, and run on 10% SDS-PAGE minigels. The proteins were transferred
to polyvinylidene difluoride (PVDF) membranes (Schleicher & Schuell,
Keene, NH) for 2 h at 100 V, and the membrane was blocked with 2%
BSA in PBS containing 1 mM EDTA, 0.05% Tween 20, and 1 mM
Na3VO4. The membrane was
probed with Ab against phosphorylated p44/42 in blocking buffer, washed
three times with 0.2% Tween 20 in 50 mM Tris (pH 8.0) and 100 mM NaCl,
then incubated with a second Ab (HRP-conjugated goat anti-rabbit
Ab) in wash buffer containing 5% nonfat dry milk. Phosphorylated bands
were visualized using the enhanced chemiluminescence system (ECL,
Amersham, Arlington Heights, IL). Immunoblotting was also conducted
using anti-phosphotyrosine 4G10, anti-Raf-1, and
anti-PKC Ab. The HRP-conjugated sheep anti-mouse Ab served
as a second Ab for anti-phosphotyrosine, anti-Raf-1, and
anti-PKC. For these experiments PMN were treated as described in
the section, Cell fractionation for immunoblotting.
Immunoprecipitation
The PMN (4 x 106/sample) were lysed in lysis buffer containing 1% Triton X-100 along with 50 mM Tris (pH 8.0), 100 mM NaCl, 1 mM Na3VO4, 1 mM PMSF, 10 µg/ml soybean trypsin inhibitor, and 1 µg/ml each of leupeptin, aprotinin, and pepstatin. Lysates were precleared with protein A-Sepharose for 30 min and incubated overnight at 4°C with anti-Syk, anti-Hck, or anti-Fgr Ab. Protein A-Sepharose was added to each sample and incubated for 2 h with rotation at 4°C. The beads were washed briefly three times with lysis buffer and twice with buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, and 1 mM Na3VO4. Adsorbed proteins were solubilized in sample buffer and separated on 10% SDS-PAGE minigels. Transfer to PVDF and subsequent immunoblotting with 4G10 anti-phosphotyrosine Ab were conducted as described above. The PVDF membranes were stripped with 100 mM 2-ME, 2% SDS, and 62.5 mM Tris (pH 6.5) at 50°C and reprobed with the appropriate Ab to demonstrate equivalent amounts of immunoprecipitated protein.
Syk kinase assay
Phagocytosing PMN were lysed and immunoprecipitated as described
in the previous section. Immunoprecipitates were washed three times
with lysis buffer, then three times with kinase buffer (50 mM HEPES (pH
7.5), 5 mM MnCl2, 2 mM
MgCl2, 10 µM
Na3VO4, and 1 mM
4-nitrophenyl phosphate). Beads were suspended in kinase buffer
containing 2 µM ATP and 10 µCi
[-32P]ATP, and Syk autophosphorylation was
conducted for 10 min at 30°C. Samples were then combined with sample
buffer, boiled 5 min, then run on 7.5% SDS-PAGE. Gels were stained,
destained, and dried, and autoradiography was performed.
The PI 3-kinase activity assay
The PMN (8 x 106/sample) were lysed
in 800 µl of a buffer containing 25 mM HEPES (pH 7.5), 50 mM NaCl, 5
mM EDTA, 1% Nonidet P-40, 1 mM
Na3VO4, and 40 mM
4-nitrophenyl phosphate. Lysates were precleared with protein
A-Sepharose for 30 min, and lysates were incubated with anti-PI
3-kinase Ab overnight with rotation at 4°C. Protein A-Sepharose was
added to each sample and incubated for 2 h with rotation at 4°C.
The beads were washed three times with the lysis buffer and three times
with kinase buffer containing 100 mM NaCl, 20 mM Tris (pH 7.5), and 1
mM EDTA. Beads were incubated in kinase buffer containing 200 µg/ml
of sonicated phosphatidylinositol substrate for 10 min at 20°C
followed by a 30-min incubation at 20°C with the addition of 2 mM
MgCl2, 2 µM ATP and 5 µCi/sample
[-32P]ATP in a total volume of 52 µl. The
reaction was terminated by adding HCl (1 M), and each sample was mixed
with chloroform and methanol to obtain a chloroform/methanol/HCl ratio
of 100/200/2. The samples were transferred to clean glass tubes and
washed with equal volumes of 1 M NaCl/chloroform/methanol (1/4/4).
After vortexing and centrifugation, 2 ml of 1% HCl was added to the
lower phase, vortexed, and centrifuged again. The lower phase was
transferred to a clean tube and dried under N2.
Lipids were spotted in a thin line on silica plates and separated using
a solvent system of chloroform/methanol/water/ammonium hydroxide
(43/38/7/5, v/v). Phosphatidylinositide 4-kinase, whose
Rf value is the same as PI-3P in this
system, was spotted as a standard and visualized using primulin.
Phosphorylated substrate was visualized by autoradiography, and the
bands scraped and counted in a liquid scintillation counter (Wallac,
Gaithersburg, MD). Activity was expressed as a percentage of the
control value.
Phospholipase D activity
The PMN were resuspended at 1 x 107/ml in PBS and labeled with 1-O-[3H]octadecyl-sn-glycero-3-phosphocholine (10-8 M; Amersham) for 30 min at 37°C. The labeled cells were washed with PBS and resuspended at 2 x 106 cells/ml in PBS containing 1 mM Ca2+ and 1 mM Mg2+. Cells were incubated with piceatannol for 30 min at 22°C, then with 200 mM ethanol for 5 min at 37°C. the PMN were then stimulated at 37°C with EIgG for 15 min or with 5 µg/ml cytochalasin B for 5 min, followed by 100 nM FMLP for 30 s. Lipids were extracted according to the method of Van Veldhoven and Bell (31), and assays for 3H-labeled phosphatidylethanol and phosphatidic acid were performed as previously described (32).
Cell fractionation for immunoblotting with PKC and Raf-1
For fractionation studies phagocytosis was stopped as described previously at 3 min after initiating the ingestion of EIgG (21). The PMN were resuspended at 1 x 108/ml in extraction buffer (50 mM Tris (pH 7.5), 2 mM EGTA, 1 mM PMSF, leupeptin (1 µg/ml), 10 µM benzamidine, 10 µM pepstatin, and aprotinin (0.2 µg/ml)). The cells were disrupted by sonication on ice, and the resulting homogenate was centrifuged (400 x g, 10 min, 4°C) to remove unbroken cells and nuclei. The supernatant of each sample was applied to a 15–40% discontinuous sucrose gradient and centrifuged for 30 min at 150,000 x g at 4°C to obtain cytosolic, membrane, and granule fractions (21). The cytosol was removed from the top of the gradient, and the membrane fraction was collected at the 15–40% interface. The granule fraction was seen as the pellet. Protein was measured by the bicinchoninic acid method (Pierce, Rockford, IL), using BSA as a standard. The cytosol and the membrane fraction were combined with sample buffer and boiled for 5 min.
Statistical analysis
Two-tailed Student’s t tests were used to assess statistical significance.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Both Syk and PI 3-kinase are implicated in mediating
FcR-mediated cell activation (9, 33, 34, 35). To determine
whether these proteins are involved in FcR-mediated phagocytosis in
PMN, we employed selective inhibitors against Syk (piceatannol) and PI
3-kinase (wortmannin) and evaluated EIgG-mediated phagocytosis by PMN.
Both piceatannol (Fig. 1
A) and
wortmannin (Fig. 1B) incubated with PMN inhibited
phagocytosis in a concentration-dependent manner. At 100 µM
piceatannol and 100 nM wortmannin, EIgG-mediated phagocytosis was
inhibited by 98 and 95%, respectively (Fig. 1, A and
B). At 100 µM, LY294002, another inhibitor of PI 3-kinase,
also inhibited phagocytosis by 86.7 ± 3.0%. The
ID50 of piceatannol and wortmannin were 1 µM
and 1 nM, respectively.
|
C). To ascertain whether the
addition of DiC10 could abrogate the inhibition by piceatannol and
wortmannin of EIgG-mediated phagocytosis, PMN were preincubated with
each of the different inhibitors, and then various concentrations of
DiC10 were added. Phagocytosis was normalized in the presence of 100
µM piceatannol after the addition of 50 µM DiC10 (Fig. 1C). At higher concentrations of DiC10 (100 and 200 µM),
phagocytosis remained similar to that in untreated controls in the
presence of 100 µM piceatannol. In contrast, when PMN were incubated
with 100 nM wortmannin, the addition of all concentrations of DiC10 was
able to augment phagocytosis of EIgG, but failed to completely
normalize phagocytosis (Fig. 1C). The maximal increase was
seen at 200 µM DiC10 addition, which led to restoration of
phagocytosis from 5–46% of the control value. Similar results were
obtained with 100 µM of the PI 3-kinase inhibitor LY294002. LY294002
inhibited EIgG phagocytosis to 13.2 ± 3.0% of control value, and
200 µM DiC10 increased the phagocytic response to 67.9 ± 10%
of the control value. Similar studies were conducted using DiC8 in lieu
of DiC10 to determine whether DiC8 could reverse the inhibitory effects
of piceatannol and wortmannin on phagocytosis. Using 10 µM DiC8,
phagocytosis was restored to 77.7 ± 8.2 and 33.7 ± 1.3% of
control values in the presence of 100 µM piceatannol and 100 nM
wortmannin, respectively. These results indicate that DAG analogues
were able to normalize piceatannol and augment wortmannin inhibition of
phagocytosis in a concentration-dependent manner. Since, following EIgG stimulation, DAG can be generated from phosphatidic acid in a pathway activated initially by phospholipase D (PLD), PLD activity was measured in PMN that were preincubated with piceatannol as another means of determining the sequence of signaling events. The 3- to 4-fold increase in PLD stimulated by phagocytosis was inhibited by piceatannol in a dose-dependent manner: 30, 61, and 99% inhibition for 1, 10, and 100 µM, respectively. In contrast, PLD stimulated by FMLP in cytochalasin B-treated PMN increased by 2-fold at 30 s and was not inhibited by 100 µM piceatannol. This supports the idea that PLD is activated by Syk during phagocytic signaling.
Protein tyrosine phosphorylation and kinase activity of Syk increase during phagocytosis of EIgG in PMN
Tyrosine phosphorylation is one of the earliest responses in PMN
activation and is required for FcR-mediated phagocytosis by
macrophages (6, 11). To evaluate whether enhanced Syk
phosphorylation occurred during ingestion of EIgG in PMN, we performed
anti-phosphotyrosine immunoblotting on lysates of phagocytosing
PMN. There was no evidence of tyrosine phosphorylation of Syk at time
zero, which was obtained by lysing PMN just before EIgG were added.
Thirty percent of maximal Syk phosphorylation was apparent within
15–30 s after initiating phagocytosis of EIgG (Fig. 2A). Phosphorylation of Syk
peaked (arbitrarily assigned 100% on autoradiographs by densitometry)
at 3–5 min, corresponding to the maximal rate of ingestion of EIgG by
PMN. Syk phosphorylation decreased by 10 min (Fig. 2A).
Blots were reprobed with mAb against Syk to demonstrate equal amounts
of Syk in immunoprecipitated samples (Fig. 2B). The
phosphorylation of Syk was an EIgG-dependent event; in contrast to
phagocytosis, activation of PMN with FMLP did not lead to
phosphorylation of Syk (data not shown). We also tested Syk kinase
activity by monitoring autophosphorylation. Syk autophosphorylation
increased to a maximum in the first few minutes of phagocytosis and
decreased by 10 min (Fig. 2C).
|
A). Syk phosphorylation was
not quite as sensitive to piceatannol as was phagocytosis, which was
inhibited 80% at 10 µM (Fig. 1A). Fig. 3B
shows that the amount of Syk was approximately equal among the samples
shown in Fig. 3A. To demonstrate the specificity of
piceatannol, we tested its effect on other kinases. The protein
tyrosine kinases Hck and Fgr have also been shown to activate upon PMN
FcR ligation (36). The PMN were preincubated with 10 or
100 µM piceatannol, as in Syk analyses, then stimulated with 100 nM
FMLP and lysed. Hck and Fgr were immunoprecipitated, and samples were
subjected to Western blotting for phosphotyrosine. Piceatannol up to
100 µM did not inhibit the FMLP-stimulated increase in
phosphorylation of Hck or Fgr (Fig. 3, C and D),
demonstrating that piceatannol is selective for Syk among these
tyrosine kinases implicated in phagocytosis. These data suggest a
direct relationship between the extent of phosphorylation of Syk and
phagocytosis of EIgG in PMN.
|
To determine whether PI 3-kinase activity could be demonstrated in PMN undergoing phagocytosis, we monitored the kinetics of PI 3-kinase in PMN challenged with EIgG. For these studies PMN were incubated with EIgG, the incubation was terminated at different time points, and the cells were lysed. The lysate was incubated with an Ab against the 85-kDa regulatory subunit of PI 3-kinase, and the resulting immunoprecipitates were tested for PI 3-kinase activity using phosphatidylinositol as the substrate, yielding PI-3P as the product.
The PI 3-kinase activity was detectable at 30 s (Fig. 4A), which correlated with the
onset of Syk phosphorylation. On the other hand, the peak of Syk
phosphorylation correlated with the maximal rate of phagocytosis,
whereas PI 3-kinase activity occurred earlier, before the maximal rate
of phagocytosis. At 30 s we observed an activity of 337 ±
47% (mean ± SEM; n = 3; kinase activity in
unstimulated PMN, 100%). Deactivation occurred at 1 min (159 ±
79%) and reached basal levels at 3 min (122 ± 13%).
|
B).
Piceatannol (condition E) inhibited PI 3-kinase activity to 10 ±
8% of the control value (condition C, Fig. 4B). In
contrast, PI 3-kinase activity of G-CSF-primed PMN stimulated by FMLP
was not inhibited by piceatannol (data not shown), indicating that PI
3-kinase itself was not directly affected by piceatannol. To
demonstrate whether PI 3-kinase was downstream from Syk activation and
to determine whether DAG could restore phagocytosis by bypassing the
inhibition of Syk and PI 3-kinase, PMN were first incubated with 100
µM piceatannol. Following the addition of 200 µM DiC10, PI 3-kinase
activity was evaluated (Fig. 4B). In the presence of 100
µM piceatannol and DiC10, PI 3-kinase activity was 16% ± 9 (Fig. 4B, condition F). Therefore, DiC10 was not able to abrogate
the piceatannol-mediated inhibition of PI 3-kinase activity, suggesting
that PLD is in a signaling pathway separate from PI 3-kinase. DiC10 did
not significantly affect PI 3-kinase activity in phagocytosing PMN
(Fig. 4B, condition D) or unstimulated PMN (condition B);
the latter condition is comparable to that in unstimulated controls
(condition A). The amount of PI 3-kinase immunoprecipitated was
unaffected by DiC10 or inhibitors and was similar between treatments
(Fig. 4C).
Phagocytosis-stimulated PI 3-kinase activity and phosphorylation of Syk
were evaluated in the presence of wortmannin. The increased activity of
PI 3-kinase following the addition of EIgG by 30 s was inhibited
by 100 nM wortmannin to 12 ± 6% (Fig. 5A). As shown in Fig. 5A, wortmannin-mediated inhibition of PI 3-kinase activity
during phagocytosis of EIgG in PMN was similar to the inhibition
mediated by piceatannol. The PI 3-kinase activity of PMN treated with
piceatannol or wortmannin was negligible and similar regardless of
whether EIgG were present (data not shown). Phosphorylation of Syk
appeared to precede the activation of PI 3-kinase, because both
wortmannin and piceatannol inhibited PI 3-kinase, but only piceatannol,
and not wortmannin, inhibited Syk (Fig. 5B). Fig. 5C shows equal amounts of immunoprecipitated Syk in all
samples. Similar to the wortmannin results, the PI 3-kinase inhibitor
LY294002 inhibited PI 3-kinase activity (by about 90%), but not
phosphorylation of Syk (data not shown). This observation suggests that
PI 3-kinase activation lies downstream of Syk in the signaling cascade
and that the signaling pathway is disrupted in piceatannol-incubated
PMN at the level of Syk activation.
|
-receptor-mediated phagocytosis in PMN
Previously, we correlated ERK1 and ERK2 phosphorylation with the
engagement of FcRII (17), demonstrated the kinetics of
ERK1 and ERK2 phosphorylation, and observed maximal phosphorylation
occurring after EIgG challenge at 5 min in PMN. To explore the upstream
signaling events leading to ERK1 and ERK2 phosphorylation during
EIgG-mediated phagocytosis, we investigated the effect of piceatannol
on ERK1 and ERK2 activation using an Ab that recognizes the
phosphorylated forms of both ERK1 and ERK2, which have molecular masses
of 44 and 42 kDa, respectively. ERK1 and ERK2 phosphorylation was
suppressed by piceatannol in a concentration-dependent manner (>80%
inhibition at 100 µM and 50% inhibition at 50 µM; Fig. 6A). Fig. 6B
demonstrates equal loading of p42/44 in all lanes. ERK phosphorylation
was not as sensitive to piceatannol as were phagocytosis or Syk
phosphorylation. Decreased sensitivity to piceatannol may occur because
ERK1 and ERK2 can be activated by reactive oxygen intermediates
(37), which are generated by PMN rendered
nonphagocytic but capable of generating reactive oxygen
intermediates through the p38 kinase pathway following engagement of
the Fc receptor (38).
|
C) (21). Addition of DiC10 to unstimulated PMN
modestly increased ERK1/2 phosphorylation to about 25% that occurring
during phagocytosis (data not shown). PMN were incubated initially with
100 µM piceatannol followed by 200 µM DiC10, then were allowed to
ingest EIgG. An increase to the phagocytosis-stimulated control value
in ERK1 and ERK2 phosphorylation was observed (Fig. 6C). The
difference between DiC10 samples with and without piceatannol was not
significant. By adding the synthetic inhibitor piceatannol to PMN, we have been able to demonstrate the effect of blocking ERK1 and ERK2 signaling downstream of Syk activation. Furthermore, these results indicate that DiC10 was able to restore ERK1 and ERK2 phosphorylation and to improve phagocytosis when piceatannol was present.
Effect of piceatannol on translocation of PKC and Raf-1 to PMN
plasma membrane during phagocytosis
We recently reported that PKC translocation is an upstream
event leading to Raf-1 translocation to the plasma membrane during
phagocytosis of EIgG in PMN and that PKC is a key component of the
phagocytic pathway (21). In turn, Raf-1 activation leads
to MEK activation followed by ERK phosphorylation (21). To
provide a basis for linking PKC to the activation of Syk, we
investigated the translocation of PKC and Raf-1 to the plasma
membrane during phagocytosis in the presence of piceatannol.
In our previous studies we found that PKC and Raf-1 were present in
the cytosol and were translocated to the plasma membrane at 3 min
following the initiation of phagocytosis (21) (Fig. 7, A and B,
condition B). Similar to the findings of others, <100% of the total
PKC and Raf-1 content of PMN was recovered from the cytosol and
membrane fraction of EIgG-stimulated cells compared with that of
control unstimulated PMN (condition A) (21, 39). These
findings suggest that these enzymes may undergo proteolysis or
translocation to other subcellular fractions (39).
Piceatannol (100 µM) inhibited both PKC and Raf-1 translocation to
the plasma membrane following 3 min of phagocytosis (Fig. 7, condition D).
|
, A and B). In our previous studies we found
that DiC10 alone led to increased translocation of PKC to the plasma
membrane during phagocytosis of EIgG (21) (Fig. 7, condition C). When PMN were preincubated with 100 µM piceatannol
followed by the addition of 200 µM DiC10, an increase in
translocation of PKC and Raf-1 to the plasma membrane occurred
during phagocytosis of EIgG (Fig. 7, condition E) compared with that in
PMN treated with piceatannol alone (condition D). These results
demonstrate that DiC10 was able to restore PKC and Raf-1
translocation during phagocytosis of EIgG when piceatannol was present.
The ability of DiC10 to restore PKC and Raf-1 translocation
correlated with the restoration of phagocytosis and ERK1 and ERK2
activation in the presence of piceatannol.
In contrast to piceatannol, there was no inhibition of PKC
translocation to the plasma membrane in the presence of wortmannin,
indicating that this PI 3-kinase inhibitor blocked phagocytosis of EIgG
in PMN in a PKC-independent process and therefore did not lead to
inhibition of ERK1 and ERK2 phosphorylation (data not shown).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
receptor engagement in PMN by analyzing protein kinase activation
during phagocytosis of EIgG. We previously found that FcR engagement
and associated signal transduction steps lead to PKC and Raf-1
translocation and, in turn, this ultimately results in activation and
phosphorylation of ERK1 and ERK2 (21). The upstream
signaling events leading to translocation of PKC and Raf-1 during
EIgG-mediated phagocytosis have not been completely elucidated. We
evaluated the role of Syk in Fc receptor-mediated signaling events
and the downstream events by examining translocation of PKC and
Raf-1 to the membrane, ERK1 and ERK2 phosphorylation, and activation of
PI 3-kinase during EIgG-mediated phagocytosis.
Several studies have shown that cross-linking Fc receptors results
in increased tyrosine phosphorylation and enzymatic activation of Syk
(11, 40, 41). We also found that EIgG is a potent stimulus
for the phosphorylation of Syk in PMN. Syk phosphorylation was maximal
at 3–5 min during phagocytosis of EIgG, which corresponded to the
maximal rate of ingested EIgG by PMN. Kinase activity of Syk followed
similar kinetics. Because piceatannol inhibits phagocytosis and Syk
activation, the mechanism by which piceatannol blocked activation of
key components was studied. The translocation of PKC and Raf-1
during EIgG-mediated phagocytosis in PMN followed by the activation of
ERK1 and ERK2 were inhibited by piceatannol, indicating that these
events are downstream of Syk phosphorylation. Therefore, Syk initiates
a cascade of protein phosphorylation during Fc receptor-mediated
phagocytosis of EIgG in PMN and provides a basis linking receptor
engagement to the signaling events mediated by ERK activation.
Phagocytosis of EIgG in PMN is associated with an increase in the
formation of DAG mass (21), which is largely generated in
a pathway initiated by the activity of PLD (42). We found
that engagement of the Fc receptor leads to activation of PLD and
the generation of phosphatidic acid. Phosphatidic acid, in turn, is
metabolized by phosphatidic phosphohydrolase to diglyceride. We
previously observed that inhibition of diglyceride generation from the
PLD pathway inhibited phagocytosis. Therefore, in these studies we
determined whether exogenous diglyceride could restore the phagocytosis
mediated by the Syk inhibitor piceatannol. We observed that in the
presence of piceatannol, DiC10 reconstituted the ERK phosphorylation
initiated by PKC and Raf-1 translocation without affecting Syk
phosphorylation. Also, PLD activity during PMN phagocytosis was
inhibited by piceatannol. These findings support the hypothesis that
the latter kinases and PLD are downstream of Syk activation. Similar to
the findings of others, we also demonstrated that EIgG ingestion by PMN
activated PI 3-kinase maximally at 30 s and that wortmannin and
LY294002, inhibitors of PI 3-kinase, prevented EIgG-stimulated
phagocytosis.
Syk activation was required for PI 3-kinase activity, because PI
3-kinase activity was inhibited by piceatannol. In contrast to the
observation with piceatannol, neither wortmannin nor LY294002 inhibited
Syk phosphorylation in PMN ingesting EIgG. Our findings extend the
observation that FcR engagement in murine macrophages induced an
increase in PI 3-kinase phosphorylation, but not in genetically
engineered Syk-deficient macrophages (34). Therefore, Syk
activation is probably required to modulate PI 3-kinase activity in
cells with Fc receptors. Chacko et al. (35) have
provided evidence that Syk phosphorylation might act as an adapter to
recruit PI 3-kinase to activated FcRII in platelets. Both Syk and PI
3-kinase activities rise significantly after only 30 s of
phagocytosis, indicating that their position is early in the signaling
cascade. The PI 3-kinase activity is transient, returning to baseline
more rapidly than that of Syk.
On the other hand, our findings do not support the hypothesis that
PKC and Raf-1 translocation and subsequent ERK1 and ERK2
phosphorylation lie downstream of PI 3-kinase activation in mediating
the PMN phagocytic response. We found that 100 nM wortmannin inhibited
phagocytosis without affecting PKC and Raf-1 translocation or ERK1
and ERK2 phosphorylation, inconsistent with PI 3-kinase regulation. The
involvement of PI 3-kinase in mediating the MAP kinase cascade is
controversial (18, 43, 44). It has been suggested that
wortmannin inhibition of the MAP kinase pathway is cell type and ligand
specific (23). Our findings are consistent with the
hypothesis that PI 3-kinase may feed into the phagocytic pathway
independently of MAP kinase activation. In accordance with our data,
Araki et al. (45) have shown that PI 3-kinase is necessary
for phagocytosis in macrophages and that PI 3-kinase contributes to the
formation of phagosomes, probably due to the closure of pseudopodia to
form intracellular vesicles. Because diglyceride is known to be
fusogenic (46, 47), this might be an explanation of why
diglyceride augments phagocytosis in wortmannin-treated PMN.
Based on these data, we propose the following model, as illustrated in
Fig. 8, for Fc receptor signaling
during PMN phagocytosis. In summary, our results demonstrated that
phagocytosis of EIgG leads to phosphorylation of Syk in PMN and that
Syk activation is required to initiate a process involving PKC,
Raf-1, ERK1, and ERK2. Furthermore, we suggest a role for PI 3-kinase
in early signal transduction events after Fc receptor-mediated
phagocytosis in PMN, but not direct involvement in the MAP kinase
cascade. It is likely that PI 3-kinase may regulate phagocytosis by a
separate and parallel pathway.
|
|
Footnotes |
|---|
2 E.M.B.R. and P.J.M. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Laurence A. Boxer, Department of Pediatrics Hematology/Oncology, University of Michigan, L2110 Women’s Hospital, Box 0238, 1500 East Medical Center Drive, Ann Arbor, MI 48109. E-mail address:
4 Abbreviations used in this paper: PMN, polymorphonuclear leukocytes; EIgG, IgG-opsonized erythrocytes; PKC, protein kinase C; ERK, extracellular signal-regulated protein kinase; PI 3-kinase, phosphatidylinositide 3-kinase; DAG, diacylglycerol; DiC8, sn-1,2-dioctanoylglycerol; DiC10, sn-1,2-didecanoylglycerol; SH2, SRC homology 2; ITAMs, immunoreceptor tyrosine-based activation motifs; MAP, mitogen-activated protein; PVDF, polyvinylidene difluoride.
Received for publication January 19, 1999. Accepted for publication October 7, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
receptor mediated phagocytosis. Blood 86:4389. receptors of phagocytes. J. Lab. Clin. Med. 126:330.[Medline]
RIIa. Biochim. Biophys. Acta 1357:348.[Medline]
receptor signaling in macrophages and neutrophils. Mol. Cell. Biol. 18:4209./Ig ß immunoreceptor tyrosine activation motif binding and autophosphorylation. J. Biol. Chem. 270:11590. subunit of Fc receptors, p72syk, and paxillin during Fc receptor-mediated phagocytosis in macrophages. J. Biol. Chem. 269:3897. receptor (CD64) induces phagocytosis in the absence of its cytoplasmic domain: the gamma subunit of Fc RIIIA imparts phagocytic function to FcRI. Exp. Hematol. 22:599.[Medline]
RII, in the absence of other Fc receptors, mediates a phagocytic signal. J. Clin. Invest. 88:1766.
subunit are required for a phagocytic signal mediated by Fc RIIIA. J. Clin. Invest. 92:2073.
receptor IIA-mediated phagocytic signal by stem-loop Syk antisense oligonucleotides. Mol. Biol. Cell 7:1095.[Abstract]
receptor cross-linking in human neutrophils. Biochem. J. 323:87.
receptors on macrophages. J. Exp. Med. 186:1027. receptor on human platelets through an immunoreceptor tyrosine-based activation motif: reconstitution with synthetic phosphopeptides. J. Biol. Chem. 271:10775. receptor signaling. J. Biol. Chem. 269:22732.RII or FcRIIIB ligation in human polymorphonuclear leukocytes. J. Biol. Chem. 270:13553. receptor I (FcRI) and receptor II (FcRII) on monocytic cells activates a signal transduction pathway common to both Fc receptors that involves the stimulation of p72 Syk protein tyrosine kinase. J. Biol. Chem. 268:24442. receptor signaling. J. Biol. Chem. 268:15900.Rs in human neutrophils. J. Leukocyte Biol. 53:427.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  J. Ennaciri and D. Girard IL-4R{alpha}, a New Member that Associates with Syk Kinase: Implication in IL-4-Induced Human Neutrophil Functions J. Immunol., October 15, 2009; 183(8): 5261 - 5269. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Jongstra-Bilen, A. Puig Cano, M. Hasija, H. Xiao, C. I. E. Smith, and M. I. Cybulsky Dual Functions of Bruton's Tyrosine Kinase and Tec Kinase during Fc{gamma} Receptor-Induced Signaling and Phagocytosis J. Immunol., July 1, 2008; 181(1): 288 - 298. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Tirouvanziam, Y. Gernez, C. K. Conrad, R. B. Moss, I. Schrijver, C. E. Dunn, Z. A. Davies, L. A. Herzenberg, and L. A. Herzenberg Profound functional and signaling changes in viable inflammatory neutrophils homing to cystic fibrosis airways PNAS, March 18, 2008; 105(11): 4335 - 4339. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Lau, X. Wang, L. Song, M. North, S. Wiehler, D. Proud, and C.-W. Chow Syk Associates with Clathrin and Mediates Phosphatidylinositol 3-Kinase Activation during Human Rhinovirus Internalization J. Immunol., January 15, 2008; 180(2): 870 - 880. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. F. Hussain, L.-Y. Kong, J. Jordan, C. Conrad, T. Madden, I. Fokt, W. Priebe, and A. B. Heimberger A Novel Small Molecule Inhibitor of Signal Transducers and Activators of Transcription 3 Reverses Immune Tolerance in Malignant Glioma Patients Cancer Res., October 15, 2007; 67(20): 9630 - 9636. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Busetto, E. Trevisan, E. Decleva, P. Dri, and R. Menegazzi Chloride Movements in Human Neutrophils during Phagocytosis: Characterization and Relationship to Granule Release J. Immunol., September 15, 2007; 179(6): 4110 - 4124. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. A. Yaghini, F. Li, and K. U. Malik Expression and Mechanism of Spleen Tyrosine Kinase Activation by Angiotensin II and Its Implication in Protein Synthesis in Rat Vascular Smooth Muscle Cells J. Biol. Chem., June 8, 2007; 282(23): 16878 - 16890. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Ratthe, M. Pelletier, S. Chiasson, and D. Girard Molecular mechanisms involved in interleukin-4-induced human neutrophils: expression and regulation of suppressor of cytokine signaling J. Leukoc. Biol., May 1, 2007; 81(5): 1287 - 1296. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Leeansyah, B. D. Wines, S. M. Crowe, and A. Jaworowski The Mechanism Underlying Defective Fc{gamma} Receptor-Mediated Phagocytosis by HIV-1-Infected Human Monocyte-Derived Macrophages J. Immunol., January 15, 2007; 178(2): 1096 - 1104. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Leseux, S. M. Hamdi, T. al Saati, F. Capilla, C. Recher, G. Laurent, and C. Bezombes Syk-dependent mTOR activation in follicular lymphoma cells Blood, December 15, 2006; 108(13): 4156 - 4162. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-S. Cha, D. L. Boyle, T. Inoue, R. Schoot, P. P. Tak, P. Pine, and G. S. Firestein A Novel Spleen Tyrosine Kinase Inhibitor Blocks c-Jun N-Terminal Kinase-Mediated Gene Expression in Synoviocytes J. Pharmacol. Exp. Ther., May 1, 2006; 317(2): 571 - 578. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. D. Moon, C. B. Post, D. L. Durden, Q. Zhou, P. De, M. L. Harrison, and R. L. Geahlen Molecular Basis for a Direct Interaction between the Syk Protein-tyrosine Kinase and Phosphoinositide 3-Kinase J. Biol. Chem., January 14, 2005; 280(2): 1543 - 1551. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Humar, N. Andriopoulos, S. E. Pischke, T. Loop, R. Schmidt, A. Hoetzel, M. Roesslein, H. L. Pahl, K. K. Geiger, and B. H. J. Pannen Inhibition of Activator Protein 1 by Barbiturates Is Mediated by Differential Effects on Mitogen-Activated Protein Kinases and the Small G Proteins Ras and Rac-1 J. Pharmacol. Exp. Ther., December 1, 2004; 311(3): 1232 - 1240. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Ryckman, C. Gilbert, R. de Medicis, A. Lussier, K. Vandal, and P. A. Tessier Monosodium urate monohydrate crystals induce the release of the proinflammatory protein S100A8/A9 from neutrophils J. Leukoc. Biol., August 1, 2004; 76(2): 433 - 440. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Ratthe and D. Girard Interleukin-15 enhances human neutrophil phagocytosis by a Syk-dependent mechanism: importance of the IL-15R{alpha} chain J. Leukoc. Biol., July 1, 2004; 76(1): 162 - 168. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Shiratsuchi and M. D. Basson Extracellular pressure stimulates macrophage phagocytosis by inhibiting a pathway involving FAK and ERK Am J Physiol Cell Physiol, June 1, 2004; 286(6): C1358 - C1366. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Krupa, H. Kato, M. A. Matthay, and A. K. Kurdowska Proinflammatory activity of anti-IL-8 autoantibody:IL-8 complexes in alveolar edema fluid from patients with acute lung injury Am J Physiol Lung Cell Mol Physiol, June 1, 2004; 286(6): L1105 - L1113. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. G. Arndt, N. Suzuki, N. J. Avdi, K. C. Malcolm, and G. S. Worthen Lipopolysaccharide-induced c-Jun NH2-terminal Kinase Activation in Human Neutrophils: ROLE OF PHOSPHATIDYLINOSITOL 3-KINASE AND Syk-MEDIATED PATHWAYS J. Biol. Chem., March 19, 2004; 279(12): 10883 - 10891. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Mansfield, S. S. Carey, V. Hinkovska-Galcheva, J. A. Shayman, and L. A. Boxer Ceramide inhibition of phospholipase D and its relationship to RhoA and ARF1 translocation in GTP{gamma}S-stimulated polymorphonuclear leukocytes Blood, March 15, 2004; 103(6): 2363 - 2368. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Hewins, J. M. Williams, M. J.O. Wakelam, and C. O.S. Savage Activation of Syk in Neutrophils by Antineutrophil Cytoplasm Antibodies Occurs via Fc{gamma} Receptors and CD18 J. Am. Soc. Nephrol., March 1, 2004; 15(3): 796 - 808. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Garcia-Garcia and C. Rosales Signal transduction during Fc receptor-mediated phagocytosis J. Leukoc. Biol., December 1, 2002; 72(6): 1092 - 1108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Barabe, E. Rollet-Labelle, C. Gilbert, M. J. G. Fernandes, S. N. Naccache, and P. H. Naccache Early Events in the Activation of Fc{gamma}RIIA in Human Neutrophils: Stimulated Insolubilization, Translocation to Detergent-Resistant Domains, and Degradation of Fc{gamma}RIIA J. Immunol., April 15, 2002; 168(8): 4042 - 4049. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Mansfield, V. Hinkovska-Galcheva, S. S. Carey, J. A. Shayman, and L. A. Boxer Regulation of polymorphonuclear leukocyte degranulation and oxidant production by ceramide through inhibition of phospholipase D Blood, February 15, 2002; 99(4): 1434 - 1441. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Chi, S. Tridandapani, W. Zhong, K. M. Coggeshall, and R. F. Mortensen C-Reactive Protein Induces Signaling Through Fc{gamma}RIIa on HL-60 Granulocytes J. Immunol., February 1, 2002; 168(3): 1413 - 1418. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Bulanova, V. Budagian, T. Pohl, H. Krause, H. Durkop, R. Paus, and S. Bulfone-Paus The IL-15R{alpha} Chain Signals Through Association with Syk in Human B Cells J. Immunol., December 1, 2001; 167(11): 6292 - 6302. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Fernandez, V. Sarma, E. Younkin, R. B. Hirschl, P. A. Ward, and J. G. Younger Exposure to perflubron is associated with decreased Syk phosphorylation in human neutrophils J Appl Physiol, November 1, 2001; 91(5): 1941 - 1947. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Desaulniers, M. Fernandes, C. Gilbert, S. G. Bourgoin, and P. H. Naccache Crystal-induced neutrophil activation. VII. Involvement of Syk in the responses to monosodium urate crystals J. Leukoc. Biol., October 1, 2001; 70(4): 659 - 668. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. S. Cooney, H. Phee, A. Jacob, and K. M. Coggeshall Signal Transduction by Human-Restricted Fc{{gamma}}RIIa Involves Three Distinct Cytoplasmic Kinase Families Leading to Phagocytosis J. Immunol., July 15, 2001; 167(2): 844 - 854. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Bharadwaj, C. Mold, E. Markham, and T. W. Du Clos Serum Amyloid P Component Binds to Fc{{gamma}} Receptors and Opsonizes Particles for Phagocytosis J. Immunol., June 1, 2001; 166(11): 6735 - 6741. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Mansfield, J. A. Shayman, and L. A. Boxer Regulation of polymorphonuclear leukocyte phagocytosis by myosin light chain kinase after activation of mitogen-activated protein kinase Blood, April 1, 2000; 95(7): 2407 - 2412. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
